Primitive progenitor cells from bone marrow are useful targets for cell-based therapies due to their self-renewing potential, multilineage differentiation, and demonstrable contribution to somatic tissues (Fellari et al., Science, 279:1528, 1998). Prevailing dogma defines three histogenetically distinct cellular systems in the bone marrow: hematopoietic cells, endothelial cells, and stromal cells, yet subsumes no common precursors in post-natal mammals (Waller et al., Blood, 85:242, 1995). Hematopoietic stem cells have been widely studied, and their lineage diagram and differentiation pathways have been defined by a number of cell surface markers as the progenitor cells differentiate into erythroid, myeloid, and lymphoid phenotypes (Bertolini et al., Exp Hematol, 24:350, 1997). Hematopoietic stem cells can be purified by flow cytochemistry using monoclonal antibodies, Hoechst 33342 and Rhodamine 123, and can be maintained as non-adherent cells in long-term bone marrow cultures in the presence of cytokines and growth factors. Conversely, bone marrow stromal cells make up the adherent cell layer in long-term in vitro bone marrow cultures and consists of cells of mesenchymal origin that generate cell lines giving rise to fibrous-osteogenic tissues of the skeleton, as well as stromal tissues which support the hematopoietic microenvironment. Marrow stromal cells, operationally called mesenchymal stem cells (MSC) can be isolated by density gradient centrifugation and adherence properties, and exhibit considerable phenotypic plasticity, including fibrogenic, osteogenic, chondrogenic, and adipogenic potential (Pittenger et al., Science, 284:143, 1999). Presumed to serve as an emergency reserve in vivo for crisis situations, the multipotentiality of MSC may also be exploited to therapeutic advantage in the development of autologous cell-based therapies and/or ex vivo gene therapy.
An alternative method has been developed to isolate mesenchymal progenitor cells under stringent survival conditions (Gordon et al., Hum. Gene. Ther., 8:1385, 1997). This technology involves the culture of bone marrow-derived cells on collagen matrices or gels impregnated with a genetically engineered growth factor, i.e., a TGFβ fusion protein bearing an auxiliary collagen-binding domain, under low serum conditions. Interestingly, the binding of TGFβ1 to collagen matrices enhanced its biologic half-life, thus permitting the isolation and expansion of TGFβ1-responsive mesenchymal progenitor cells. This physiological response to the TGFβ1 is both necessary and sufficient for the capture (i.e., survival) of these blastoid cells, which are otherwise not physically separated from either hematopoietic or other mesenchymal cells on the basis of size, density, adherence properties, or cell surface markers. The TGFβ1-responsive cells proliferate readily upon serum reconstitution, and form distinctive colonies within the TGFβ1-vWF impregnated collagen gel. The morphology of these cells is initially blastoid, spherical and non-adherent, not fibroblastic, yet the proliferative cells were capable of overt cytodifferentiation into fibroblastic, chondrogenic and/or osteogenic cells, signifying a mesenchymal precursor. When placed in bone chambers in a subcutaneous rat model, the TGFβ1-responsive mesenchymal progenitor cells formed cartilage in vivo, as well as bone. In contrast, the bone morphogenic protein (BMP)-captured stem cells exhibited a less proliferative and more differentiated osteogenic phenotype in vivo (Andrades et al, Exp. Cell Res., 250:485, 1997).
There is a need for the identification and isolation of progenitor cells capable of giving rise to mesenchymal or hematopoietic stem cells. Further, there is a need for a method of identifying these progenitor cells when present in a population of cells. The identification, and methods for achieving identification, of such cells will have considerable implications for cell biology and gene therapy protocols.